Lafora disease (LD) is an intractable fatal epilepsy with onset in teenage years. Our groups across this project discovered the genetic causes and disease mechanisms of LD. The LD genes, EPM2A and EPM2B, encode respectively the laforin glycogen phosphatase and the malin ubiquitin E3 ligase, which regulate glycogen synthesis, in particular its structure. The pathogenic pathology is the neuronal accumulation of poorly branched and insoluble glycogen, termed polyglucosan, into massive inclusions, Lafora bodies (LB), which drive the epilepsy. Polyglucosans, though malformed glycogen, are glycogen nonetheless, and only one enzyme generates glycogen strands, namely glycogen synthase (GS). We showed in LD mouse models that 50-100% elimination of GS activity through GS knockout experiments rescues LD. Several projects in this proposal leverage this discovery to devise a comprehensive approach towards therapy, ranging from genome editing of the GS gene to small molecule inhibition of the enzyme. It is anticipated that inhibiting glycogen synthesis will prevent further LB formation and stop disease progression. Since these patients are no sicker than regular epilepsy cases early in their course, stopping the disease will be tantamount to cure. But what about cases already in the throes of intractable seizures? It is not known whether LB are straight accumulations versus a net accumulation where clearance mechanisms are overwhelmed by the constant formation of new polyglucosans. If the latter is the case, inhibiting further polyglucosan generation may lead to disease reversal, i.e. would be effective even in advanced cases. In Aim 1, we generate a mouse model in which the glycogen synthase 1 gene (Gys1), the muscle and brain isoform of GS, is deleted at various time points of the disease course in which LBs have already formed, and we analyze the progression or reversal of the phenotype histologically and by electrophysiological and behavioral experiments. In Aim 2, we generate a mouse model in which only one allele of the Gys1 gene is deleted at various time points in which LBs have already formed to check whether a 50% inhibition would be sufficient to prevent/revert the progression of this condition. These two aims will specify the window of opportunity for therapeutic intervention in LD for therapies aimed at glycogen synthesis inhibition. In Aim 3 we answer another unknown, namely whether reintroducing the actual missing genes (EPM2A or EPM2B) in LD can arrest the disease, or reverse it. We generate an Epm2b-/- LD mouse in which we activate Epm2b expression in different phases of the disease to determine whether this can stop disease, and whether it can reverse it. The results of this project will be crucial knowledge towards future human therapy.